5`S-LNA nucleotides and oligonucleotides

ABSTRACT

The invention relates to a compound of formula (I) wherein R2 and R4 are joined and together form a group, such a —CH2O—. The compound of formula (I) can be used in the manufacture of 5&#39;S-LNA oligonucleotides as antisense drugs.

The invention relates to a LNA phosphoramidite building block bearing asulfur atom in the 5′-position of the sugar ring, to a process for themanufacture of this building block, and to a process for manufacturingan oligonucleotide comprising this building block.

The invention thus relates in particular to a compound of formula (I)

-   -   wherein    -   R¹ is a phosphate protecting group;    -   R² and R⁴ together form —CH₂O—, —CH₂NH—, —CH₂S—, —CH₂N(OR^(p))—,        —CHCH₃O—, —C(CH₃)₂O—, —CH₂C(═CH₂)—, —CHCH₃C(═CH₂)—, —CHCH₃S—,        —CH₂NR^(p)—, —CH₂CH₂O—, —CH₂CH₂CH₂O—, —CH₂OCH₂—, —CH(CH₂OCH₃)O—,        —CH(CH₂CH₃)O— or —CH₂OCH₂O—;    -   R³ is dialkylamino;    -   R⁵ is a thiohydroxyl protecting group;    -   each R^(p) is alkyl; and    -   Nu is a nucleobase optionally comprising a protected primary        amino group.

The use of oligodeoxynucleotides as therapeutic agents, where thewell-understood principles of Watson-Crick hybridization are exploitedto target complementary RNA strands, has witnessed remarkable progresssince its inception in the late 1970's (P. C. Zamecnik, M. L.Stephenson, P Natl Acad Sci USA 1978, 75, 280-284; S. T. Crooke,Antisense drug technology: principles, strategies, and applications, 2nded. ed., Boca Raton, Fla.: CRC Press, 2008).

Several types of chemical modifications have been introduced over timein synthetic oligonucleotides in order to e.g. extend their half-life,improve pharmacokinetics, enhance the RNaseH activity, reduce toxicityor enhance mismatch discrimination.

One of the most successful modifications is the introduction ofphosphorothioate linkages, where one of the nonbridging phosphate oxygenatoms is replaced with a sulfur atom (F. Eckstein, Antisense and NucleicAcid Drug Development 2009, 10, 117-121). Such phosphorothioateoligodeoxynucleotides show an increased protein binding as well as adistinctly higher stability to nucleolytic degradation and thus asubstantially higher half-life in plasma, tissues and cells than theirunmodified phosphodiester analogues. This allowed the development of thefirst generation of oligonucleotide therapeutics and opened the door ofthe later generation modifications such as Locked Nucleic Acids (LNAs).

Replacement of a phosphodiester linkage with a phosphorothioate,however, creates a chiral center at the phosphorous atom. As aconsequence, all approved oligonucleotide therapeutics are until nowmixtures of a huge amount of diastereoisomeric compounds, withpotentially different (and possibly opposing) physiochemical properties.

In order to reduce the diastereoisomeric complexity of sucholigodeoxynucleotides, the sulfur atom within a phosphorothioate can intheory be shifted from one of the nonbridging positions to the bridging5′-position of the ribose sugar. This modification renders thesubstitution pattern around the phosphorous symmetrical and thus removesthe chiral center, consequently reducing the diastereoisomericcomplexity of the molecule.

Oligonucleotides containing such 2′,5′-dideoxy-5-mercapto buildingblocks have been prepared (see R. Cosstick, J. Gaynor, Curr. Org. Chem.2008, 12, 291-308 and references therein) and examined in diversecontexts such as for example site specific cleavage (e.g. K.Jahn-Hofmann, J. W. Engels, Helv. Chim. Acta 2004, 87, 2812-2828) ormechanistic investigations in chemical biology (e.g. S. R. Das, J. A.Piccirilli, Nat Chem Biol 2005, 1, 45-52). Their application in atherapeutic context especially with the intent of reducing thediastereoisomeric complexity of phosphorothioate oligonucleotides,however, has received far less attention. Furthermore, the combinationof such a 2′,5′-dideoxy-5′-mercapto modification with a Locked NucleicAcid (LNA) sugar moiety is completely unprecedented.

We have surprisingly found a process that gave access for the first timeto LNA phosphoramidite building blocks bearing a sulfur atom in the5′-position of the sugar ring and applied them to the synthesis ofmodified oligonucleotides. The invention thus provides an entry intosuch 5′-mercapto modified LNAs. When included into oligonucleotides, itreduces the diastereomeric complexity of the resulting molecule.

In the present description the term “alkyl”, alone or in combination,signifies a straight-chain or branched-chain alkyl group with 1 to 8carbon atoms, particularly a straight or branched-chain alkyl group with1 to 6 carbon atoms and more particularly a straight or branched-chainalkyl group with 1 to 3 carbon atoms. Examples of straight-chain andbranched-chain C₁-C₈ alkyl groups are methyl, ethyl, propyl, isopropyl,butyl, isobutyl, tert.-butyl, the isomeric pentyls, the isomeric hexyls,the isomeric heptyls and the isomeric octyls, particularly methyl,ethyl, propyl, butyl and pentyl more particularly methyl, ethyl, propyl,isopropyl, isobutyl, tert.-butyl and isopentyl. Particular examples ofalkyl are methyl, ethyl, propyl and isopropyl.

The term “alkoxy” or “alkyloxy”, alone or in combination, signifies agroup of the formula alkyl-O— in which the term “alkyl” has thepreviously given significance, such as methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, isobutoxy, sec.butoxy and tert.butoxy. Particular“alkoxy” are methoxy and ethoxy.

The term “halogen” or “halo”, alone or in combination, signifiesfluorine, chlorine, bromine or iodine and particularly chlorine, bromineor iodine. The term “halo”, in combination with another group, denotesthe substitution of said group with at least one halogen, particularlysubstituted with one to five halogens, particularly one to fourhalogens, i.e. one, two, three or four halogens.

The term “aryl”, alone or in combination, signifies the phenyl ornaphthyl group.

The terms “hydroxyl” and “hydroxy”, alone or in combination, signify the—OH group.

The terms “thiohydroxyl” and “thiohydroxy”, alone or in combination,signify the —SH group.

The term “amino”, alone or in combination, signifies the primary aminogroup (—NH₂), the secondary amino group (—NH—), or the tertiary aminogroup (—N—).

The term “oxy”, alone or in combination, signifies the —O— group.

The term “protecting group”, alone or in combination, signifies a groupintroduced into a molecule by chemical modification of a functionalgroup to obtain chemoselectivity in a subsequent chemical reaction.

“Phosphate protecting group” is a protecting group of the phosphategroup. Examples of phosphate protecting group are 2-cyanoethyl andmethyl. A particular example of phosphate protecting group is2-cyanoethyl.

“Hydroxyl protecting group” is a protecting group of the hydroxyl group.Examples of hydroxyl protecting groups are acetyl (Ac), benzoyl (Bz),benzyl (Bn), β-methoxyethoxymethyl ether (MEM), dimethoxytrityl (orbis-(4-methoxyphenyl)phenylmethyl) (DMT), trimethoxytrityl (ortris-(4-methoxyphenyl)phenylmethyl) (TMT), methoxymethyl ether (MOM),methoxytrityl [(4-methoxyphenyl)diphenylmethyl (MMT), p-methoxybenzylether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl(THP), tetrahydrofuran (THF), trityl or triphenylmethyl (Tr), silylether (for example trimethylsilyl (TMS), tert-butyldimethylsilyl(TBDMS), tri-iso-propylsilyloxymethyl (TOM) and triisopropylsilyl (TIPS)ethers), methyl ethers and ethoxyethyl ethers (EE). Particular examplesof hydroxyl protecting group are DMT and TMT, in particular DMT.

“Thiohydroxyl protecting group” is a protecting group of thethiohydroxyl group. Examples of thiohydroxyl protecting groups are thoseof the “hydroxyl protecting group”.

The term “leaving group” refers to a molecular fragment that can departwith a pair of electrons in heterolytic bond cleavage. Examples ofleaving group are Cl, Br, I, OTs, OTf or OMs.

The term “nucleophile” refers to a chemical species capable of donatingan electron pair to another species, during a reaction, to form achemical bond.

The term “nucleobase” refers to the base moiety of a nucleotide andcovers both naturally occurring a well as non-naturally occurringvariants. Thus, “nucleobase” covers not only the known purine andpyrimidine heterocycles but also heterocyclic analogues and tautomeresthereof. Examples of nucleobases include, but are not limited toadenine, guanine, cytosine, thymidine, uracil, xanthine, hypoxanthine,5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurineand 2-chloro-6-aminopurine.

The term “nucleotide” as used herein, refers to a glycoside comprising asugar moiety, a base moiety and a covalently linked group (linkagegroup), such as a phosphate or phosphorothioate internucleotide linkagegroup, and covers both naturally occurring nucleotides, such as DNA orRNA, and non-naturally occurring nucleotides comprising modified sugarand/or base moieties.

The term “solid support” refers to supports used for the solid phasesynthesis, in particular of oligomeric compounds. Examples of solidphase support comprise crosslinked polystyrene (Primer Support 5G orNittoPhaseHL), controlled pore glass (CPG); oxalyl-controlled poreglass, silica-containing particles, such as porous glass beads andsilica gel such as that formed by the reaction oftrichloro-[3-(4-chloromethyl)phenyl]propylsilane and porous glass beads(PORASIL E®). Controlled pore glass is a particular useful solid phasesupport.

The term “oligonucleotide synthesis activator” refers to a compoundcapable of activating the reaction of an unprotected nucleoside with anincoming nucleoside phosphoramidite monomer. Examples of sucholigonucleotide synthesis activators can be found in X. Wei, Tetrahedron2013, 69, 3615-3637. Examples of oligonucleotide synthesis activatorsare azole based activators like 1H-tetrazole, 5-nitrophenyl-1H-tetrazole(NPT), 5-ethylthio-1H-tetrazole (ETT), 5-benzylthio-1H-tetrazole (BTT),5-methylthio-1H-tetrazole (MTT), 5-mercapto-tetrazoles (MCT) and4,5-dicyanoimidazole (DCI), or acidic salts like pyridiniumhydrochloride, imidazoliuim triflate, benzimidazolium triflate,5-nitrobenzimidazolium triflate, or weak acids such as2,4-dinitrobenzoic acid or 2,4-dinitrophenol.5-(3,5-bis(trifluoromethyl)phenyl)-1H-tetrazole is a particularly usefuloligonucleotide synthesis activator.

The term “capping” or “capping step” refers to the conversion ofhydroxyl or thiohydroxyl groups that have not reacted during theoligonucleotide coupling into a protected hydroxyl or thiohydroxylgroup. The capping thus hinders the reaction of said hydroxyl orthiohydroxyl groups in the next coupling steps. The capping step is forexample conveniently performed by the reaction with acetic anhydride(Ac₂O) or phenoxyacetic anhydride (Pac-anhydride), for example incombination with activators like pyridine and N-methyl-imidazole, forexample in THF or acetonitrile. The resulting protected hydroxyl orthiohydroxyl group is for example an acetate or thioacetate group.

The term “sugar modified nucleoside” refers to a nucleoside wherein thesugar is other than DNA or RNA.

The invention thus relates in particular to:

A compound of formula (I) wherein R¹ is cyanoethyl or methyl;

A compound of formula (I) wherein R² and R⁴ together form —CH₂O—;

A compound of formula (I) wherein R³ is diisopropylamino;

A compound of formula (I) wherein R⁵ is trityl, 4-methoxytrityl,4,4′-dimethoxytrityl or 4,4′,4″-trimethoxytrityl;

A compound of formula (I) wherein R⁵ is 4,4′-dimethoxytrityl;

A compound of formula (I) wherein each Rp is independently methyl, ethylor propyl;

A compound of formula (I) wherein the nucleobase is adenine, guanine,cytosine, 5-methyl-cytosine, thymine or uracil;

A compound of formula (I) wherein Nu is (A), (B), (C) or (D)

wherein R⁶ is hydrogen or a protecting group of the amino group to whichit is attached;

A compound of formula (I) wherein the protecting group of the aminogroup is benzoyl, dimethylformamide, acetyl or isobutyryl; and

A compound of formula (I) wherein Nu is (A1), (B1) or (C1)

The invention further relates to a compound of formula (I) selected from

-   N-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide;-   3-[({[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-3-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl]oxy}[bis(propan-2-yl)amino]phosphanyl)oxy]propanenitrile;-   N′-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide;    and-   N-{1-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide.

The invention further relates in particular to a compound selected from

-   N-(9-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-9H-purin-6-yl)benzamide;-   N-{9-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide;-   N-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide;-   1-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione;-   N′-(9-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylmethanimidamide;-   N′-{9-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide;-   N′-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide;-   N-(1-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide;-   N-{1-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide    and;-   N-{1-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide.

The synthesis of the compound of formula (I) can be made for exampleaccording to the following schemes. Unless otherwise specified, R¹ to R⁹and Nu have the meaning as defined above.

Starting from a 5′-protected nucleoside 1 (e.g. 5′-DMT) as well as baseprotected LNA building blocks (e.g. Nu=protected A, G or C), thecorresponding LNA 5′/3′ diols 2 are obtained by an acid promoteddeprotection. The sulfur is introduced into the 5′ position in 3 bymeans of a Mitsunobu reaction using e.g. thiobenzoic acid as thenucleophile. Subsequent hydrolysis of the resulting thioester gives thefree thiol 4, which is then protected with e.g. a DMT group to arrive at5. Finally, the desired phosphoramidite building block (I) ready forsolid phase oligonucleotide synthesis is obtained by phosphitylation ofthe 3′-hydroxyl group using a suitable phosphoramidite together with atetrazole derivative as an acidic activator.

A LNA 5′/3′ diol 1 (e.g. Nu=T) is converted to the corresponding5′-mesylate 2 by the treatment with e.g. methanesulfonyl chloride in thepresence of e.g. DMAP in pyridine. This intermediate is then reactedwith a protected thioacetate (e.g. DMT-protected thioacetate, obtainedby the reaction of e.g. DMT chloride with thioacetic acid) in thepresence of base and e.g. sodium methoxide as a nucleophile to arrive at3. The resulting product is then converted to the desiredphosphoramidite building block (I) by phosphitylation of the 3′-hydroxylgroup with a phosphoramidite in the presence of an acidic tetrazoleactivator.

The invention thus also relates to:

A process for the manufacture of a compound of formula (I) comprisingthe reaction of a compound of formula (II)

in the presence of P(R³)₂OR¹ and an oligonucleotide synthesis activatorwherein R¹ to R⁵ and Nu are as defined above;

A process according to the invention wherein the compound of formula(II) as defined above is obtained by the reaction of a compound offormula (III)

in the presence of R⁵X¹ and a base, wherein R², R⁴, R⁵ and Nu are asdefined above and wherein X¹ is a leaving group;

A process according to the invention wherein X¹ is Cl, Br, I, OTs, OTfor OMs;

A process according to the invention wherein the compound of formula(III) as defined above is obtained by the hydrolysis of a compound offormula (IV)

wherein

-   -   R⁷ is alkyl, aryl, arylalkyl, substituted aryl or substituted        arylalkyl, wherein substituted aryl and substituted arylalkyl        are aryl and arylalkyl substituted on the aryl with one to three        substitutents independently selected from alkyl, alkoxy and        halogen; and

wherein R², R⁴ and Nu are as defined above;

A process according to the invention wherein the hydrolysis of acompound of formula (IV) is done in the presence of a nucleophile, likee.g. NaOH, KOH, NaOMe, KOMe, methylamine or NH₃, in particular NaOH;

A process according to the invention wherein substituted aryl andsubstituted arylalkyl are aryl and arylalkyl substituted on the arylwith one to three substitutents independently selected from methyl,methoxy, chlorine, bromine and iodine;

A process according to the invention wherein R⁷ is methyl, ethyl,propyl, isopropyl, benzyl, phenyl, substituted benzyl or substitutedphenyl, wherein substituted phenyl and substituted benzyl are phenyl andbenzyl substituted on the phenyl with one to three substitutentsindependently selected from methyl, methoxy, chlorine, bromine andiodide;

A process according to the invention wherein R⁷ is phenyl;

A process according to the invention wherein the compound of formula(IV) as defined above is obtained by the reaction of a compound offormula (V)

in the presence of R⁷COSH, a phosphine and a dehydrating agent, whereinR², R⁴, R⁷ and Nu are as defined above;

A process according to the invention wherein the compound of formula (V)is obtained by the removal of the hydroxyl protecting group R⁵ of acompound of formula (VI)

wherein R⁵ is a hydroxyl protecting group, and wherein R², R⁴ and Nu areas defined above;

A process according to the invention wherein the compound of formula(II) as defined above is obtained by the reaction of a compound offormula (VII)

in the presence of a base, a nucleophile and a compound of formula(VIII)

wherein

R⁸ is a leaving group;

R⁹ is alkyl; and

R², R⁴, R⁵ and Nu are as defined above;

A process according to the invention wherein R⁹ is methyl;

A process according to the invention wherein the compound of formula(VII) as defined above is obtained by the reaction of a compound offormula (V) as defined above in the presence of R⁸X² and anon-nucleophilic base, wherein X² is a leaving group and R⁸ is a groupcapable of forming a leaving group together with the 5′ hydroxyl oxygenatom of the compound of formula (V);

A process according to the invention wherein X² is Cl, Br, I, OTs, OTfor OMs;

A process according to the invention wherein the compound of formula(VIII) as defined above is obtained by the reaction of R⁵X³ in thepresence of R⁹C(O)SH wherein R⁵ and R⁹ are as defined above and X³ is aleaving group;

A process according to the invention wherein X³ is Cl, Br, I, OTs, OTfor OMs;

A process according to the invention wherein the oligonucleotidesynthesis activator is an azole;

A process according to the invention wherein the oligonucleotidesynthesis activator is 1H-tetrazole, 5-nitrophenyl-1H-tetrazole (NPT),5-ethylthio-1H-tetrazole (ETT), 5-benzylthio-1H-tetrazole (BTT),5-methylthio-1H-tetrazole (MTT), 5-mercapto-tetrazoles (MCT) or4,5-dicyanoimidazole (DCI);

A process according to the invention wherein the nucleophile used toobtain the compound of formula (II) as defined above is NaOH, KOH,NaOMe, KOMe, methylamine or NH₃;

A process according to the invention wherein the nucleophile used toobtain the compound of formula (II) as defined above is NaOMe;

A process according to the invention wherein the dehydrating agent isdiethyl azodicarboxylate or diisopropyl azodicarboxylate;

A process according to the invention wherein the dehydrating agent isdiethyl azodicarboxylate;

A process according to the invention wherein the phosphine istriphenylphosphine or trimethylphosphine;

A process according to the invention wherein the phosphine istriphenylphosphine;

A process according to the invention wherein the removal of the hydroxylprotecting group R⁵ of a compound of formula (VI) as defined above isdone by the reaction of a compound of formula (VI) in the presence ofacid;

A process according to the invention wherein the acid is perchloroaceticacid, acetic acid, chloroacetic acid, dichloroacetic acid ortrifluoroacetic acid;

A process according to the invention wherein the acid is perchloroaceticacid;

A compound of formula (II) as defined above;

A compound of formula (III) as defined above; and

A compound of formula (IV) as defined above.

The invention further relates to the use of a compound of the inventionfor the manufacture of an oligonucleotide.

The invention is also directed to an oligonucleotide comprising afragment of formula (IX)

wherein R², R⁴ and Nu are as defined above.

The invention also relates to a method for the manufacture of anoligonucleotide comprising a fragment of formula (IX) as defined abovecomprising the following steps:

-   -   (a) Providing a solid support comprising:        -   a hydroxyl group;        -   a nucleotide comprising a hydroxyl group; or        -   an oligonucleotide comprising a hydroxyl group;    -   (b) Coupling a compound of the invention, a nucleotide, a locked        nucleic acid nucleotide, a 2′-sugar modified nucleotide, a        3'S-DNA or a 3'S-LNA to the hydroxyl group of said solid        support;    -   (c) Oxidizing or thiooxydizing the product obtained from (b);    -   (d) Optionally capping unreacted hydroxyl groups of the product        obtained from step (c);    -   (e) Optionally removing hydroxyl protecting groups or        thiohydroxyl protecting groups from the product obtained from        step (c) or (d);    -   (f) Optionally repeating steps (b) to (e);    -   (g) Optionally removing any remaining protecting groups from the        product obtained from any one of steps (c) to (f); and    -   (h) Optionally cleaving the oligonucleotide from the solid        support.

The invention relates in particular to a method for the manufacture ofan oligonucleotide comprising a fragment of formula (IX) according tothe invention, comprising coupling at least one compound of theinvention in a step (b), and in particular a compound of formula (I).

The invention will now be illustrated by the following examples whichhave no limiting character.

EXAMPLES Abbreviations:

AcOH=acetic acid, CAS RN=chemical abstracts registration number,DMAP=4-dimethylaminopyridine, DME=dimethoxyethane,DMF=N,N-dimethylformamide, DIPEA=N,N-diisopropylethylamine, dppf=1,1bis(diphenylphosphino)ferrocene, EI=electron impact, ESI=electrosprayionization, h=hour,HATU=1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxidehexafluorophosphate, HPLC=high performance liquid chromatography,ISP=ion spray positive (mode), ISN=ion spray negative (mode),min=minutes, LAH=lithium aluminium hydride, LiHMDS=lithiumbis(trimethylsilyl)amide, MPLC=medium performance liquid chromatography,MS=mass spectrum, PG=protecting group, Pd-C=palladium on activatedcarbon,PdCl₂(dppf)-CH₂Cl₂=1,1′-bis(diphenylphosphino)ferrocene-palladium(II)dichloridedichloromethane complex, RT=room temperature,S-PHOS=2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl,TFA=trifluroacetic acid, THF=tetrahydrofuran,TMEDA=N,N,N′,N′-tetramethylethylenediamine,HBTU=O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate,DMEDA=N,N′-dimethylethylenediamine, ACN=acetonitrile, TBAI=tetra butylammonium iodile, DME=di methoxy ethane, DEAD=diethyl azodicarboxylate,DMTrCl=4,4′-dimethoxytrityl chloride.All examples and intermediates were prepared under argon atmosphere ifnot specified otherwise.

Example 1N-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide

A solution of 5-ethylmercapto-1H-tetrazole (1.3 g, 9.97 mmol, 0.25 Msolution in 38.4 mL dry ACN CAS RN 89797-68-2) was added to a stirredsolution of N-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide(3.5 g, 4.99 mmol) in dry DCM (120 mL) under argon at room temperaturefollowed by addition of 2-cyanoethyl tetraisopropylphosphorodiamodite(3.17 mL, 9.98 mmol, CAS RN 102691-36-1). The reaction mixture wasstirred at room temperature for 4 h. Then the reaction mixture wasdiluted with DCM (300 mL) and poured onto a sat. NaHCO₃ solution (100mL). The organic layer was separated off and the aqueous layer wasextracted with DCM (70 mL×2). The combined organic layer was dried oversodium sulfate, filtered and evaporated under reduced pressure. Theresulting crude compound was purified by combiflash (10-20% ACN in DCM)to get (2.7 g) impure compound. Using the same protocol another 1 gbatch was performed to get 0.6 g impure compound. Using the sameprotocol another 2.5 g were carried out to get 1 g (pure) compound and1.5 g impure compound. The impure compound thus obtained from differentbatches was mixed and repurified to get the title product (3.0 g) whichwas mixed with pure compound (1 g) to get the title compound (4.0 g,44%) as a white solid. MS: (ESI): m/z=901.6 [M+H]⁺.

Example 1.1N-{9-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide

To a solution of Cl₃CCOOH (2.98 g, 18.23 mmol, CAS RN 76-03-9) in DCM(150 ml) was addedN-[9-(1-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-9H-purin-6-yl]benzamide(10 g, 14.58 mmol) at 25° C. Then the reaction mixture was stirred for 3h at 25° C. Volatiles were removed under reduced pressure and theresulting crude was purified by combiflash (10% MeOH in DCM) to get thetitle product (2) (5 g, 89%) as a white solid. MS: (ESI): m/z=383.8[M+H]⁺.

Example 1.2N-(9-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-9H-purin-6-yl)benzamide

To an ice cooled solution of PPh₃ (10.26 g, 39.13 mmol CAS RN 603-35-0)in anhydrous THF (150.0 mL) was added DEAD (6.14 mL, 39.13 mmol, CAS RN1972-28-7) and the reaction mixture was stirred at 0° C. for 30 min.PhCOSH (4.62 mL, 39.13 mmol, CAS RN 98-91-9) was added drop-wise to thereaction mixture and it was stirred at 0° C. for another 30 min.N-{9-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide(5.0 g, 13.04 mmol) was added to the stirred reaction mixture at 0° C.for 2 h. The reaction mixture was then stirred at room temperature for 2h. The reaction mixture was diluted with water (200 mL) and extractedwith ethyl acetate (120 mL×3). The combined organic part was washed withNaHCO₃ (100 mL), dried over Na₂SO₄, filtered and evaporated underreduced pressure to get the title product (25 g, crude) as a yellowviscous oil. MS: (ESI): m/z=504.3 [M+H]⁺.

Example 1.3N-{9-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide

Argon was bubbled through NaOH (0.5 M, 238 mL) as well as solution ofTHF-MeOH (6:4, 250 mL) for 30 min.N-(9-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-9H-purin-6-yl)benzamide(20 g, crude) was dissolved in an argon purged solution of THF-MeOH(6:4, 250 mL) under argon and cooled at 0° C. to −5° C. To this solutionwas added NaOH solution (0.5 M, 238 mL, 119.16 mmol) and the reactionmixture was stirred at 0° to −5° C. for 30 min. Then a solution ofcitric acid (30.04 g, 142.98 mmol) was added at 0° C. A saturated NaHCO₃solution (300 mL) was added to the reaction mixture and the mixture wasextracted with ethyl acetate (200 mL×3). The combined organic layer waswashed with brine, dried over sodium sulfate, filtered and evaporatedunder reduced pressure to get the title product (20 g, crude) as an offwhite viscous oil. MS: (ESI): m/z=400.2 [M+H]⁺.

Example 1.4N-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide

To a solution ofN-{9-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide(20 g, crude) in anhydrous pyridine (20 mL, argon purged) was addedDMTrCl (5.09 g, 15.02 mmol CAS RN 40615-36-9) at 25° C. and the reactionmixture was stirred at 25° C. for 4 h. Volatiles were removed underreduced pressure and the reaction mixture was diluted with DCM (300 mL).The DCM layer was washed with a NaHCO₃ solution (100 mL×2) followed bybrine (100 mL). The organic layer was dried over Na₂SO₄, filtered andevaporated under reduced pressure. The resulting crude was purified bycombiflash (2% MeOH in DCM containing 0.5% TEA) to get the title product(5 g, 68% over 3 steps) as a pale yellow solid. MS: (ESI): m/z=702.14[M+H]⁺.

Example 23-[({[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-3-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl]oxy}[bis(propan-2-yl)amino]phosphanyl)oxy]propanenitrile

A solution of 5-ethylmercapto-1H-tetrazole (1.77 g, 13.59 mmol, 0.25 Msolution in 55.3 mL dry ACN, CAS RN 89797-68-2) and 2-cyanoethyltetraisopropylphosphorodiamodite (4.31 mL, 13.59 mmol, CAS RN102691-36-1) were added sequentially to a stirred solution of1-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione(4.0 g, 6.80 mmol) in dry DCM (150 mL) under argon at 25° C. and thereaction mixture was stirred at 25° C. for 4 h. Then the reactionmixture was diluted with DCM (200 mL) and poured onto a sat. NaHCO₃solution (200 mL). The organic layer was separated off and the aq. layerwas extracted with DCM (70 mL×2). The combined organic layer was driedover sodium sulfate, filtered and evaporated under reduced pressure. Theresulting crude compound was purified by FCC under argon atmosphere(10-20% ACN in DCM) to get title compound (TM-6) (2.45 g, 46%) as an offwhite solid. MS: (ESI): m/z=789.4 [M+H]⁺

Example 2.1[7-hydroxy-3-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-2,5-dioxabicyclo[2.2.1]heptan-1-yl]methylmethanesulfonate

To a stirred solution of1-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione(10 g, 37.00 mmol) in anhydrous pyridine (100.0 mL, CAS RN 110-86-1)were added DMAP (0.452 g, 3.7 mmol) and methane sulfonyl chloride (3.15mL, 40.71 mmol) at 0° C. to −5° C. sequentially. Then the reactionmixture was stirred at 0° C. for another 3 h. Volatiles were removedunder reduced pressure and the resulting crude was purified bycombiflash (5% MeOH in DCM) to get the title product (2) (8 g, 62%) as apale white solid. MS: (ESI): m/z=348.97 [M+H]⁺.

Example 2.21-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

To an argon purged solution of1-{[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}ethan-1-one (10.43 g,27.56 mmol) in dry DMF (80 mL) was added a freshly prepared solution ofNaOMe (prepared by adding sodium (0.758 g, 34.45 mmol) to 24 mL ofanhydrous methanol followed by the dilution of the resulting solutionwith DMF (8 mL)) in a drop-wise manner at 25° C. under argon. To theresulting reaction mixture was added a solution of[7-hydroxy-3-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-2,5-dioxabicyclo[2.2.1]heptan-1-yl]methylmethanesulfonate (4.0 g, 11.48 mmol) and 1,1,3,3 tetramethyl guanidine(3.03 mL, 24.12 mmol, CAS RN 80-70-6) in DMF (32 mL) rapidly. Thereaction mixture was stirred at 25° C. for 3 h. Then the reactionmixture was diluted with DCM (400 mL) and washed with a sat. NaHCO₃solution (200 mL). The organic layer was dried over sodium sulfate,filtered and evaporated under reduced pressure. The crude compound waspurified by combiflash (ethyl acetate containing 0.5% TEA) to get thetitle product (3.3 g, 49%) as a pale brown solid. MS: (ESI): m/z=588.89[M+H]⁺.

Example 2.3 1-{[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}ethan-1-one

To a solution of DMTrCl (10.0 g, 29.51 mmol, CAS RN 40615-36-9) inanhydrous DCM (100.0 mL) was added CH₃COSH (6.24 ml, 88.54 mmol, CAS RN507-09-5) drop-wise at 0° C. to −5° C. and the reaction mixture wasstirred at 0° C. for another 30 min. After completion of reaction, thereaction mixture was neutralized with TEA (12.2 mL, 88.54 mmol)drop-wise at 0° C. Then the reaction mixture was washed with a saturatedNaHCO₃ solution (200 mL×2) and brine (100 mL), dried over sodiumsulfate, filtered and concentrated under vacuum to get the titlecompound (11 g, 98%) as pale brown waxy solid.

Example 3N′-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide

A solution of 5-ethylmercapto-1H-tetrazole (2.65 g, 20.34, 0.25 Msolution in 87.5 mL dry ACN, CAS RN 89797-68-2) and 2-cyanoethyltetraisopropylphosphorodiamodite (6.45 mL, 20.34 mmol, CAS RN102691-36-1) were added sequentially to a stirred solution ofN′-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide(6.8 g, 10.17 mmol) in dry DCM (200 mL) at 25° C. and the reactionmixture was stirred at 25° C. for 4 h. Then the reaction mixture wasdiluted with DCM (250 mL) and poured onto a sat. NaHCO₃ solution (200mL). The organic layer was separated off and the aq. layer was extractedwith DCM (2×100 mL). The organic layer was dried over sodium sulfate,filtered and evaporated under reduced pressure. The resultant crudecompound was purified by combiflash (90% ACN in DCM) to get titlecompound (4.2 g, 48%).MS: (ESI): m/z=869.0 [M+H]⁺.

Example 3.1N′-{9-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide

To a solution of2-amino-9-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6,9-dihydro-1H-purin-6-one(10 g, 33.87 mmol) in DMF (100 mL) was added DMF-DMA (9.0 mL, 67.74mmol, CAS RN 4637-24-5) at 25° C. The mixture was stirred for 4 h at 25°C. Volatiles were removed under reduced pressure and the resultant crudecompound was washed with n-hexane (50 mL×3) and dried to get the titlecompound (13 g, crude) as white solid. MS: (ESI): m/z=350.7 [M+H]⁺.

Example 3.2N′-(9-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylmethanimidamide

To an ice cooled solution of PPh₃ (29.20 g, 111.32 mmol, CAS RN603-35-0) in anhydrous THF (400 mL) was added DEAD (17.46 mL, 111.32mmol, CAS RN 1972-28-7) and the reaction mixture was stirred at 0° C.for 30 min. PhCOSH (13.15 mL, 37.11 mmol, CAS RN 98-91-9) was addeddrop-wise to the reaction mixture and stirring continued at 0° C. foranother 30 min.N′-{9-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide(13 g, crude) was added and stiffing continued at 0° C. for 2 h. Thenthe reaction mixture was stirred at room temperature for 2 h. Volatileswere removed under reduced pressure and the resultant crude compound waspurified by combiflash (5% MeOH in DCM) get the title product (18 g,crude) as a pale yellow solid. MS: (ESI): m/z=470.83 [M+H]⁺.

Example 3.3N′-{9-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide

To an ice cooled solution ofN′-(9-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylmethanimidamide(18 g, crude) in anhydrous MeOH (300 mL) was added K₂CO₃ (42.30 g,306.05 mmol, CAS RN 584-08-7) and the reaction mixture was stirred at 0°C. for 2 h and at 25° C. for 2 h. The solvent was evaporated underreduced pressure to get the title compound (60 g, crude) as a pale brownsolid. MS: (ESI): m/z=366.6 [M+H]⁺.

Example 3.4N′-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide

To a solution ofN′-{9-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide(60 g, crude) in anhydrous pyridine (80 mL, CAS RN 110-86-1) degassedwith argon was added DMTrCl (19.42 g, 57.32 mmol, CAS RN 40615-36-9) at25° C. and the reaction mixture was stirred at 25° C. for 4 h. Volatileswere removed under reduced pressure and the crude reaction mixture wasdiluted with water (200 mL). The aq. phase was extracted with ethylacetate (3×250 mL). The combined organic part was dried over Na₂SO₄,filtered and evaporated under reduced pressure. The resultant crude waspurified by combiflash (5% MeOH in DCM containing 0.5% TEA) to get thetitle product (6.8 g, 30% over 4 steps) as a pale yellow solid. MS:(ESI): m/z=668.6 [M+H]⁺.

Example 4N-{1-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide

To an ice-cooled solution of N-{1-[1-({[bis(4-methoxyphenyl) (phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide(4.8 g, 6.938) in DCM (50 mL) was added a solution of5-ethylthio-1H-tetrazole (1.8 g, 13.87 mmol, CAS RN 89797-68-2) inacetonitrile (35 mL) followed by 2-cyanoethyl tetra isopropylphosphorodiamidite (6.6 mL, 20.18 mmol, CAS RN 102691-36-1) under argonatmosphere at 25° C. and the reaction mixture was stirred for 4 h at 25°C. Then the reaction mixture was poured onto a saturated NaHCO₃ solution(50 mL) and the organic layer was separated off. The aq. layer wasextracted with DCM (25 mL×2). The combined organic layer was dried oversodium sulfate, filtered and evaporated under reduced pressure. Theresulting crude was purified by column chromatography over silica gel(20% acetonitrile in DCM) to get the title product (3.0 g, 48% yield) asan off white solid. Using the above protocol, another 3 g batch wascarried out and mixed with this batch to get the deliverable amount. MS:(ESI): m/z=892.1 [M+H]⁺.

Example 4.1N-{1-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide

To a stirred solution of CCl₃CO₂H (4.5 g, 27.74 mmol, CAS RN 76-03-9) inDCM (100 mL) was addedN-[1-(1-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl]benzamide(15 g, 22.19 mmol) at 25° C. and the reaction mixture was stirred for 2hours at 25° C. After completion, volatiles were removed under reducedpressure to get the crude residue which was purified by combiflash (10%MeOH in DCM) to get the title product (5.8 g, 69%) as an off-whitesolid. MS: (ESI): m/z=373.9 [M+H]⁺.

Examples 4.2N-(1-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide

To an ice-cooled solution of PPh₃ (11.1 g, 42.58 mmol, CAS RN 603-35-0)in dry THF (150 mL) was added DEAD (6.7 mL, 42.58 mmol, CAS RN1972-28-7) drop-wise and the reaction mixture was stirred at 0° C. underargon atmosphere for 30 min. To this was added PhCOSH (5.03 mL, 42.58mmol, CAS RN 98-91-9) and stirring continued at 0° C. for 30 min. To theresultant reaction mixture was addedN-{1-[7-hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide(5.3 g, 14.19 mmol) and stirring continued at 0° C. for 2 h and 2 h at25° C. Then the reaction mixture was diluted with water (200 mL) andextracted with ethyl acetate (100 mL×3). The combined organic layer wasdried over Na₂SO₄ and evaporated under reduced pressure. The resultingcrude was purified by combiflash chromatography (50% ethyl acetate inhexane) to get the title product (7.2 g, crude, contaminated with PPh₃O)as an off white solid which was used as such in the next step withoutfurther purification MS: (ESI): m/z=493.6 [M+H]⁺.

Example 4.3N-{1-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide

To a degassed solution ofN-(1-{1-[)benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide(2.0 g, 4.052 mmol) in THF/MeOH (6:4) (40 mL) under argon was added asolution of NaOH (0.5 M, 24.3 mL) at −5° C. and the reaction mixture wasstirred at 0 to −5° C. for 30 min. To the resultant reaction mixture wasadded a 10% aq. solution of citric acid (30 mL) at 0° C. A saturatedsodium bicarbonate solution (50 mL) was added to the reaction mixtureand the compound was extracted with ethyl acetate (70 mL×3). Thecombined organic layer was washed with brine (100 mL×2), dried oversodium sulfate and concentrated under reduced pressure to get the titlecompound (1.7 g, crude) as a white solid which was used as such in thenext step without further purification. MS: (ESI): m/z=390.2 [M+H]⁺.

Example 4.4N-{1-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide

To an argon purged solution of crudeN-{1-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide(5.5 g, 14.123 mmol) in dry pyridine (20 mL, CAS RN 110-86-1) was addedDMTrCl (7.1 g, 21.18 mmol, CAS RN 40615-36-9) at 25° C. the reactionmixture was stirred at 25° C. for 16 h. Volatiles were removed underreduced pressure and the crude reaction mixture thus obtained wasdiluted with DCM (200 mL). The organic part was washed with a sat.NaHCO₃ solution (50 mL×2) followed by brine (50 mL×2), dried over sodiumsulfate and evaporated under reduced pressure. The resultant crude waspurified by combiflash chromatography (35% ethyl acetate in hexanecontaining 0.5% TEA) to get impure an compound which was repurified bycombiflash (30% ethyl acetate/hexane) to get the title compound (4.8 g,49% yield). MS: (ESI): m/z=690.7 [M+H]⁺.

1. A compound of formula (I)

wherein R¹ is a phosphate protecting group; R² and R⁴ together form—CH₂O—, —CH₂NH—, —CH₂S—, —CH₂N(OR′)—, —CHCH₃O—, C(CH₃)₂O, —CH₂C(═CH₂)—,—CHCH₃C(═CH₂)—, —CHCH₃S—, —CH₂NR^(p)—, —CH₂CH₂O—, —CH₂CH₂CH₂O—,—CH₂OCH₂—, —CH(CH₂OCH₃)O—, —CH(CH₂CH₃)O— or —CH₂OCH₂O—; R³ isdialkylamino; R⁵ is a thiohydroxyl protecting group; each R^(p) isalkyl; and Nu is a nucleobase optionally comprising a protected primaryamino group.
 2. A compound according to claim 1, wherein R¹ iscyanoethyl or methyl.
 3. A compound according to claim 1, wherein R² andR⁴ together form —CH₂O—.
 4. A compound according to claim 1, wherein R³is diisopropylamino.
 5. A compound according to claim 1, wherein R⁵ istrityl, 4-methoxytrityl, 4,4′-dimethoxytrityl or4,4′,4″-trimethoxytrityl.
 6. A compound according to claim 1, whereineach Rp is independently methyl, ethyl or propyl.
 7. A compoundaccording to claim 1, wherein Nu is adenine, guanine, cytosine,5-methyl-cytosine, thymine or uracil.
 8. A compound according to claim1, wherein Nu is (A), (B), (C) or (D)

wherein R⁶ is hydrogen or a protecting group of the amino group to whichit is attached.
 9. A compound according to claim 8, wherein theprotecting group of the amino group is benzoyl, dimethylformamide,acetyl or isobutyryl.
 10. A compound according to claim 1, wherein Nu is(A1), (B1) or (C1)


11. A process for the manufacture of a compound of formula (I) asdefined in claim 1, comprising the reaction of a compound of formula(II)

in the presence of P(R³)₂OR¹ and an oligonucleotide synthesis activator,wherein R¹ is a phosphate protecting group; R² and R⁴ together form—CH₂O—, —CH₂NH—, —CH₂S—, —CH₂N(OR′)—, —CHCH₃O—, C(CH₃)₂O, —CH₂C(═CH₂)—,—CHCH₃C(═CH₂)—, —CHCH₃S—, —CH₂NR^(p)—, —CH₂CH₂O—, —CH₂CH₂CH₂O—,—CH₂OCH₂—, —CH(CH₂OCH₃)O—, —CH(CH₂CH₃)O— or —CH₂OCH₂O—; R³ isdialkylamino; R⁵ is a thiohydroxyl protecting group; and each R^(p) isalkyl.
 12. A process according to claim 11, wherein the compound offormula (II) is obtained by the reaction of a compound of formula (III)

in the presence of R⁵X¹ and a base, wherein R² and R⁴ together form—CH₂O—, —CH₂NH—, —CH₂S—, —CH₂N(OR′)—, —CHCH₃O—, C(CH₃)₂O, —CH₂C(═CH₂)—,—CHCH₃C(═CH₂)—, —CHCH₃S—, —CH₂NR^(p)—, —CH₂CH₂O—, —CH₂CH₂CH₂O—,—CH₂OCH₂—, —CH(CH₂OCH₃)O—, —CH(CH₂CH₃)O— or —CH₂OCH₂O—; R³ isdialkylamino; R⁵ is a thiohydroxyl protecting group; each R^(p) isalkyl; Nu is a nucleobase optionally comprising a protected primaryamino group and wherein X¹ is a leaving group.
 13. A process accordingto claim 12, wherein the compound of formula (III) is obtained by thehydrolysis of a compound of formula (IV)

wherein R⁷ is alkyl, aryl, arylalkyl, substituted aryl or substitutedarylalkyl, wherein substituted aryl and substituted arylalkyl are aryland arylalkyl substituted on the aryl with one to three substitutentsindependently selected from alkyl, alkoxy and halogen.
 14. A processaccording to claim 13, wherein R⁷ is phenyl.
 15. A process according toclaim 13, wherein the compound of formula (IV) is obtained by thereaction of a compound of formula (V)

in the presence of R⁷COSH, a phosphine and a dehydrating agent, whereinR⁷ is alkyl, aryl, arylalkyl, substituted aryl or substituted arylalkyl,wherein substituted aryl and substituted arylalkyl are aryl andarylalkyl substituted on the aryl with one to three substitutentsindependently selected from alkyl, alkoxy and halogen.
 16. A processaccording to claim 15, wherein the compound of formula (V) is obtainedby the removal of the hydroxyl protecting group R⁵ of a compound offormula (VI)

wherein R⁵ is a hydroxyl protecting group.
 17. A process according toclaim 11, wherein the compound of formula (II) is obtained by thereaction of a compound of formula (VII)

in the presence of a base, a nucleophile and a compound of formula(VIII)

wherein R⁸ is a leaving group; R⁹ is alkyl.
 18. A process according toclaim 17, wherein the compound of formula (VII) is obtained by thereaction of a compound of formula (V)

in the presence of R⁸X² and a non-nucleophilic base, wherein X² is aleaving group and R⁸ is a group capable of forming a leaving grouptogether with the 5′ hydroxyl oxygen atom of the compound of formula(V).
 19. A process according to claim 17, wherein the compound offormula (VIII) is obtained by the reaction of R⁵X³ in the presence ofR⁹C(O)SH X³ is a leaving group.
 20. A process according to claim 11,wherein the oligonucleotide synthesis activator is an azole.
 21. Aprocess according to claim 11, wherein the oligonucleotide synthesisactivator is 1H-tetrazole, 5-nitrophenyl-1H-tetrazole (NPT),5-ethylthio-1H-tetrazole (ETT), 5-benzylthio-1H-tetrazole (BTT),5-methylthio-1H-tetrazole (MTT), 5-mercapto-tetrazoles (MCT) or4,5-dicyanoimidazole (DCI).
 22. A process according to claim 17, whereinthe nucleophile is NaOH, KOH, NaOMe, KOMe, methylamine or NH₃.
 23. Aprocess according to claim 15, wherein the dehydrating agent is diethylazodicarboxylate or diisopropyl azodicarboxylate.
 24. A processaccording to claim 12, wherein the phosphine is triphenylphosphine ortrimethylphosphine.
 25. A process according to claim 16, wherein theremoval of the hydroxyl protecting group R⁵ of a compound of formula(VI) is done by the reaction of a compound of formula (VI) in thepresence of acid.
 26. A process according to claim 25, wherein the acidis perchloroacetic acid, acetic acid, chloroacetic acid, dichloroaceticacid or trifluoroacetic acid.
 27. (canceled)
 28. (canceled) 29.(canceled)
 30. A compound according to claim 1 selected fromN-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide;3-[({[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-3-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl]oxy}[bis(propan-2-yl)amino]phosphanyl)oxy]propanenitrile;N′-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide;andN-{1-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide.31. A compound selected fromN-(9-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-9H-purin-6-yl)benzamide;N-{9-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide;N-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-9H-purin-6-yl}benzamide;1-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione;N′-(9-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylmethanimidamide;N′-{9-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide;N′-{9-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-N,N-dimethylmethanimidamide;N-(1-{1-[(benzoylsulfanyl)methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl}-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide;N-{1-[7-hydroxy-1-(sulfanylmethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamideand;N-{1-[1-({[bis(4-methoxyphenyl)(phenyl)methyl]sulfanyl}methyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-3-yl]-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide.32. (canceled)
 33. An oligonucleotide comprising a fragment of formula(IX)

wherein R² and R⁴ together form —CH₂O—, —CH₂NH—, —CH₂S—, —CH₂N(OR′)—,—CHCH₃O—, —C(CH₃)₂O—, —CH₂C(═CH₂)—, —CHCH₃C(═CH₂)—, —CHCH₃S—,—CH₂NR^(p)—, —CH₂CH₂O—, —CH₂CH₂CH₂O—, —CH₂OCH₂—, —CH(CH₂OCH₃)O—,—CH(CH₂CH₃)O— or —CH₂OCH₂O—; wherein each R′ is alkyl; and Nu is anucleobase optionally comprising a protected primary amino group.
 34. Amethod for the manufacture of an oligonucleotide comprising a fragmentof formula (IX) as defined in claim 33 comprising the following steps:(a) Providing a solid support comprising: a hydroxyl group; a nucleotidecomprising a hydroxyl group; or an oligonucleotide comprising a hydroxylgroup; (b) Coupling a compound according to claim 1, a nucleotide, alocked nucleic acid nucleotide, a 2′-sugar modified nucleotide, a3'S-DNA or a 3'S-LNA to the hydroxyl group of said solid support; (c)Oxidizing or thiooxydizing the product obtained from (b); (d) optionallycapping unreacted hydroxyl groups of the product obtained from step (c);(e) Optionally removing hydroxyl protecting groups or thiohydroxylprotecting groups from the product obtained from step (c) or (d); (f)Optionally repeating steps (b) to (e); (g) Optionally removing anyremaining protecting groups from the product obtained from any one ofsteps (c) to (f); and (h) Optionally cleaving the oligonucleotide fromthe solid support.
 35. (canceled)